Anti-Cyclin B1 抗体 [Y106]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin B1 antibody [Y106] (AB32053)

Unpurified ab32053 at a 1 : 100 dilution staining Human cyclin B1 in human skin carcinoma, using Immunohistochemistry, Paraffin Embedded Tissue.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cyclin B1 antibody [Y106] (AB32053)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cyclin B1 with Purified ab32053 at 1 : 100 dilution. Cells were fixed in 100% Methanol. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1 : 1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cyclin B1 antibody [Y106] (AB32053)

Unpurified ab32053 staining Cyclin B1 in the U2OS cell line from human by ICC/IF (Immunocytochemistry/immunofluorescence).Cells were fixed with formaldehyde,permeabilized with 1% Triton X-100 in PBS and blocked with 1% BSA for 1 hour at 37°C.Alexa Fluor® 594-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Stephanie Hilss.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cyclin B1 antibody [Y106] (AB32053)

Overlay histogram showing Jurkat cells stained with unpurified ab32053 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32053, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin B1 antibody [Y106] (AB32053)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cervical carcinoma tissue sections labeling Cyclin B1 with Purified ab32053 at 1 : 250 dilution (1.47 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cyclin B1 antibody [Y106] (AB32053)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cyclin B1 with purified ab32053 at 1/400 dilution (1μg/ml) (Right). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Left). Cells were pre-treated with 20μg/ml RNaseA for 30min to minimize the binding between PI and RNA.Then intracellular stained with ab32053 and PI.

• IP

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Immunoprecipitation - Anti-Cyclin B1 antibody [Y106] (AB32053)

ab32053 (purified) at 1 : 20 dilution (2μg) immunoprecipitating Cyclin B1 in Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate.

Lane 1 (input) : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10μg
Lane 2 (+) : ab32053 & Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32053 in Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Cyclin B1 antibody [Y106] (ab32053)

Predicted band size: 48 kDa

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• WB

Lab

Western blot - Anti-Cyclin B1 antibody [Y106] (AB32053)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : CCNB1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lanes 1 - 3 : Merged signal (red and green). Green - ab32053 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab32053 was shown to specifically react with CCNB1 in wild-type HAP1 cells. No band was observed when CCNB1 knockout samples were examined. Wild-type and CCNB1 knockout samples were subjected to SDS-PAGE. ab32053 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/3000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Cyclin B1 antibody [Y106] (ab32053)

Predicted band size: 48 kDa

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• WB

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Western blot - Anti-Cyclin B1 antibody [Y106] (AB32053)

All lanes:

Western blot - Anti-Cyclin B1 antibody [Y106] (ab32053) at 1/20000 dilution

Lane 1:

Hela cell lysate.

Lane 2:

Jurkat cell lysate.

Predicted band size: 48 kDa

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• WB

Lab

Western blot - Anti-Cyclin B1 antibody [Y106] (AB32053)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Cyclin B1 antibody [Y106] (ab32053) at 1/50000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 48 kDa

Observed band size: 58 kDa

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